Myricanol derivatives and uses thereof for treatment of neurodegenerative diseases

ABSTRACT

The subject invention pertains to myricanol derivatives, therapeutic compositions, and methods for treatment of neurodegenerative diseases, in particular, neurodegenerative diseases associated with abnormal accumulation of protein tau.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application of InternationalPatent Application No. PCT/US2013/035628, filed Apr. 8, 2013, whichclaims priority to U.S. Application No. 61/621,278, filed Apr. 6, 2012and 61/678,409, filed Aug. 1, 2012, the disclosures of each of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Intracellular aggregation of abnormal species of phosphorylated tau(protein tau) is a major pathologic feature of a family ofneurodegenerative diseases collectively referred to as the tauopathies.Tau normally functions to stabilize microtubules in neurons; however, itpathologically aggregates more than 15 neurodegenerative diseases,including Alzheimer's disease (AD) and Parkinson's disease. The mostcommon tauopathy is Alzheimer's disease, in which paired helicalfilaments (PHFs) of mis-folded protein tau aggregates in neurofibrillarytangles, in dystrophic neuritis of senile plaques, and in cell processesin the neuropil. Abnormal accumulation of protein tau is closely linkedwith postsymptomatic progression in Alzheimer's disease. Abnormalaccumulation of protein tau in the cytoplasm of neuronal and glial cellsalso represents major structural hallmarks in the pathology of Pick'sdisease, corticobasal degeneration, and progressive supranuclear palsy.

At present, researchers on the development of therapeutics fortauopathies focus primarily on agents that prevent abnormalphosphorylation or aggregation of tau proteins. However, it has beendiscovered that while aggregation of hyperphosphorylated protein tau isvisible evidence of tauopathies, these neurofibrillary tangles appear tobe less toxic than soluble intermediates of protein tau. High levels oftau intermediates, particularly aberrant tau species failed to becleared from cells, cause cognitive dysfunction in AD and tauopathies.Therefore, agents that degrade or destabilize tau intermediates, clearaberrant tau species from cells, or otherwise reduce intracellular taulevels, are promising therapeutics for AD and tauopathies.

Existing therapeutics for the treatment tauopathies (such as AD) onlydemonstrate limited efficacy. Additional therapeutics for the treatmentof tauopathies are needed.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides myricanol derivatives for treatingneurodegenerative diseases, in particular, neurodegenerative diseasesassociated with abnormal accumulation of protein tau.

In certain specific embodiments, the subject invention providesmyricanol derivatives for treating neurodegenerative diseases, whereinthe myricanol derivatives are selected from Compound 22 and Compound 23:

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence of a tau protein isoform (tau 352)useful according to the subject invention.

SEQ ID NO:2 is an amino acid sequence of a tau protein isoform (tau 441)useful according to the subject invention.

SEQ ID NO:3 is an amino acid sequence of a tau protein isoform (tau 383)useful according to the subject invention.

SEQ ID NO:4 is an amino acid sequence of a tau protein isoform (tau 758)useful according to the subject invention.

SEQ ID NO:5 is an amino acid sequence of a tau protein isoform (tau 776)useful according to the subject invention.

SEQ ID NO:6 is an amino acid sequence of a tau protein isoform (tau 412)useful according to the subject invention.

DETAILED DESCRIPTION

The subject invention provides myricanol derivatives for treatingneurodegenerative diseases, in particular, neurodegenerative diseasesassociated with abnormal accumulation of protein tau.

Compounds

In certain embodiments, the subject invention provides compounds 1-34,or salts thereof (as shown in Table 1).

Molecular Compound Structure Weight Name/sample names Results 1

358.4281 Myricanol/Myr-R-1-G/ BBtol-F/BBtol-F-E moderate activity 2

356.4123 myricanone 3

344.4016 Myricananin A 4

372.4117 Porson 5

454.5122 Myricanol triacetate 6

446.4041 4-[5′-(3-carboxypropanoyl)- 4,4′-dihydroxy-2′,6-dimethoxy[1,1′-biphenyl-3- yl]-4-oxobutanoic acid 7

374.4275 Hexahydrocurcumin 8

372.4117 Tetrahydrocurcumin 9

338.3539 Demethoxycurcumin active 10

308.3279 Bisdemethoxycurcumin active 11

350.4923 [10]-Gingerol Active 12

322.4391 [8]-Gingerol 13

294.3859 [6]-Gingerol 14

596.8369 Myricanol 11- monopalmitate/Myr-R-1-E 15

833.2729 Myricanol 11,17- dipalmitate/Myr-R-3-E-4 16

596.8369 myricanol 17- palmitate/Myr-R-3-F 17

596.8369 myricanol 5- palmitate/Myr-R-3-E-2 18

340.4129 Myricacene A (trans)/ Myr-R-9-4; Myr-16-13-4 active 19

340.4129 Myricacene B (trans)/ Myr-R-9-2-8; Myr-R-16-15 active 20

576.8488 Myricacene B (trans) 5-palmitate/Myr-R20-2 21

340.4129 Myricacene A (cis)/ Myr-R-9-2-4; Myr-16-13-2 moderately active22

340.4129 Myricacene B (cis)/ Myr-R-9-2-6 active 23

340.4129 Myr-R-16-6 very active 24

340.4129 Myr-R-16-4-2 active 25

344.4016 Myr-R-21-4-2 26

344.4016 Myr-R-21-4-4 27

330.3750 Myr-R-21-2 28

372.4117 Myr-R-25-2/Myr-R-26-2 29

464.5485 Myr-R-25-8 30

372.4117 Myr-R-27-2 31

386.4813 Dimethylmyricanol/ Myr-R-31 32

400.5079 Trimethylmyricanol/ Myr-R-33-4 33

368.4660 Dimethylmyricacene/ Myr-R-32-2 34

312.3597 Myr-R30-2

In certain embodiments, the subject invention pertains to the uses ofCompound 9, Compound 10, Compound 11, Compound 18, Compound 19, Compound21, Compound 22, Compound 23, and Compound 24, and salts thereof, fortreating neurodegenerative diseases, in particular, neurodegenerativediseases associated with abnormal accumulation of protein tau.

In certain specific embodiments, the subject invention pertains to theuses of compounds 22 and 23, or salts thereof, for treatingneurodegenerative diseases, in particular, neurodegenerative diseasesassociated with abnormal accumulation of protein tau.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula A, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula B, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula C, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula D, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula E, or a salt thereof,

wherein R₁-R₈ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula F, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula G, or a salt thereof,

wherein R₁-R₃ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula H, or a salt thereof,

wherein R₁ and R₂ are, independently, —H or any group that forms anester or ether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula I, or a salt thereof,

wherein R₁-R₃ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula J, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula K, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In one embodiment, the subject invention provides an isolated orsubstantially pure compound of formula L, or a salt thereof,

wherein R₁-R₄ are, independently, —H or any group that forms an ester orether bond.

In certain embodiments, one or more of R₁-R₈ of formula A or formula Lcan be, —H, unsubstituted or substituted alkyl, alkenyl, —COOH, acyl,benzyl, or cyclic alkyl. In certain embodiments, any or all of R₁-R₈have fewer than 6 carbon atoms.

In certain embodiments, one or more of R₁-R₈ of formula A or formula Lcan be an organic or inorganic acid group including, but not limited to,acetic acid, carboxylic acid, aspartic acid, formic acid, citric acid,benzoic acid, hippuric acid, malic acid, mucic acid, phosphoric acid,sulfuric acid, gluconic acid, maleic acid, succinic acid, tartaric acid,and lactic acid.

In certain embodiments, one or more of R₁-R₈ of formula A or formula Lcan be a carbohydrate moiety, in which a monosaccharide, disaccharide,oligosaccharide, or its derivative loses an —H in its hydroxyl group andthereby forms a radical. Suitable carbohydrate moieties can be derived,for example, from glucose, fructose, and sucrose.

The compounds of the present invention can be synthesized. In preferredembodiments, the compounds of the present invention are at least 75%pure, preferably at least 90% pure, more preferably are more than 95%pure, and most preferably are more than 99% pure (substantially pure).

As used herein, “isolated” refers to compounds that have been removedfrom any environment in which they may exist in nature.

The present invention further embodies stereoisomers of the compounds offormula A or formula L. The term “stereoisomer” encompasses allenantiomerically/stereomerically pure andenantiomerically/stereomerically enriched compounds disclosed herein.

The enantiomeric forms of the compounds (e.g., isolated or chemicallysynthesized) of the invention are substantially free from one another(i.e., in enantiomeric excess). In other words, the “+” forms of thecompounds are substantially free from the “−” forms of the compounds.Conversely, “−” forms of the compounds are substantially free of “+”forms of the compounds. In one embodiment of the invention, theenantiomeric compounds are in at least about 80% of the “+” forms. In apreferred embodiment, the compounds are in at least about 90% of the “+”forms. In a more preferred embodiment, the compounds are in at leastabout 95% of the “+” forms. In an even more preferred embodiment, thecompounds are in at least about 97.5% of the “+” forms. In a mostpreferred embodiment, the compounds are in at least about 99% of the “+”forms.

“Alkyl” means a linear saturated monovalent radical of one to sixteencarbon atoms or a branched saturated monovalent of three to sixteencarbon atoms. It may include hydrocarbon radicals of one to four or oneto three carbon atoms, which may be linear. Examples include methyl,ethyl, propyl, 2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, and thelike.

“Alkenyl” means a linear or branched C₂-C₁₆ hydrocarbon radical thatcomprises one or more carbon-carbon double bonds. Examples includepropylenyl, buten-1-yl, isobutenyl, penten-1-yl, 2,2-methylbuten-1-yl,3-methylbuten-1-yl, hexan-1-yl, hepten-1-yl, octen-1-yl, and the like.

“Acyl” means a radical —C(O)R where R is hydrogen, alkyl or cycloalkyl,or heterocycloalkyl. Examples include formyl, acetyl, ethylcarbonyl, andthe like.

“Carboxyl” means the radical —C(O)OH.

“Halo” means fluoro, chloro, bromo or iodo.

“Haloalkyl” means alkyl substituted with one or more same or differenthalo atoms, e.g., —CH₂Cl, —CH₂Br, —CF₃, —CH₂CH₂Cl, —CH₂CCl₃, and thelike.

“Hydroxy” means the radical —OH.

“Hydroxyalkyl” means an alkyl radical as defined herein, substitutedwith one or more, preferably one, two or three hydroxy groups.Representative examples include, but are not limited to, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxy-propyl, preferably 2-hydroxyethyl,2,3-dihydroxypropyl and 1-(hydroxymethyl) 2-hydroxyethyl.

“Substituted,” as used herein, refers to a compound or chemical moietyin which at least one hydrogen atom of that compound or chemical moietyis replaced with a second chemical moiety. In certain embodiments,substituents include, but are not limited to, halogen; alkyl;heteroalkyl; alkenyl; alkynyl; hydroxyl, aryl, hydroxyalkyl, heteroaryl,hydroxy; alkoxyl; amino; nitro; thiol; carbocyclic cycloalkyl, amino(primary, secondary or tertiary); o-lower alkyl; o-aryl, aryl;aryl-lower alkyl.

In one embodiment, the subject invention pertains to the therapeutic useof Compound 22, Compound 23, and Compound 24:

In one embodiment, the subject invention pertains to the therapeutic useof the following isomer of Compound 23:

Treatment of Neurodegenerative Diseases

The compounds and compositions of the subject invention, throughadministration to a subject, are useful for treating or amelioratingneurodegenerative diseases or conditions, in particular,neurodegenerative diseases or conditions associated with abnormally highlevels of protein tau and/or abnormal accumulation of protein tau inneurons. In a preferred embodiment, the compounds and compositions ofthe subject invention are useful to treat or ameliorate Alzheimer'sdisease or Parkinson's disease.

In one embodiment, the subject invention provides a method for treatinga neurodegenerative disease or condition, particularly a disease orcondition associated with abnormally high levels of protein tau and/orabnormal accumulation of protein tau in neurons, wherein the methodcomprises administering, to a subject in need of such treatment, aneffective amount of a composition comprising a compound selected fromCompound 9, Compound 10, Compound 11, Compound 18, Compound 19, Compound21, Compound 22, Compound 23, and Compound 24, or a salt thereof.

In one embodiment, the subject invention provides a method for treatinga neurodegenerative disease or condition, particularly a disease orcondition associated with abnormally high levels of protein tau and/orabnormal accumulation of protein tau in neurons, wherein the methodcomprises administering, to a subject in need of such treatment, aneffective amount of a composition comprising a compound selected fromany of formula A or formula L, or a salt thereof.

In one embodiment, the subject invention provides a method of reducingintracellular tau levels, wherein said method comprises administering,to cells comprising protein tau, an effective amount of a compound offormula A to formula L.

In one embodiment, the following isomer of Compound 23 is administeredto a subject having a neurodegenerative disease or condition,particularly a disease or condition associated with abnormally highlevels of protein tau and/or abnormal accumulation of protein tau inneurons,

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the compositionsaccording to the subject invention can be administered. Mammalianspecies that can benefit from the disclosed methods of treatmentinclude, but are not limited to, apes, chimpanzees, orangutans, humans,monkeys; and other animals such as dogs, cats, horses, cattle, pigs,sheep, goats, chickens, mice, rats, guinea pigs, and hamsters.Typically, the subject is a human.

The term “tau protein” or any grammatical variation thereof (e.g.,protein tau and tau etc.), as used herein, refers generally to anyprotein of the microtubule-associated tau protein family. Members of thetau family share the common features of a characteristic N-terminalsegment, sequences of approximately 50 amino acids inserted in theN-terminal segment, a characteristic tandem repeat region consisting of3 or 4 tandem repeats of 31-32 amino acids, and a C-terminal tail. Tauproteins of the subject invention may be in a form of soluble tauintermediates, functional, aberrant, abnormally-truncated, mis-folded ormis-processed tau, and phosphorylated tau.

Preferably, tau protein of the subject invention is of mammalian origin,more preferably, of human origin. Specifically, tau proteins of thesubject invention include microtubule-associated protein translated fromthe human chromosomal sequence of GenBank Accession No. AH005895 andnaturally-occurring mammalian variants or isoforms thereof. Six humanbrain tau isoforms are currently known, including tau352 (GenBankAccession No. NP 058525) (SEQ ID NO:1), tau441 (GenBank Accession No. NP005901) (SEQ ID NO:2), tau383 (GenBank Accession No. NP 058518) (SEQ IDNO:3), tau758 (GenBank Accession No. NP 058519) (SEQ ID NO:4), tau776(GenBank Accession No. NP 001116538) (SEQ ID NO:5), and tau412 (GenBankAccession No. NP 001116539) (SEQ ID NO:6).

The term “treatment” or any grammatical variation thereof (e.g., treat,treating and treatment etc.), as used herein, includes but is notlimited to, ameliorating or alleviating a symptom of a disease orcondition, reducing, suppressing, inhibiting, lessening, or affectingthe progression, severity, and/or scope of a condition, chance ofre-occurrence or returning of a disease after a remission. For instance,the term “treatment” includes (i) ameliorating a symptom associated witha neurodegenerative disease in a patient diagnosed with theneurodegenerative disease; and/or (ii) relieving (such as attenuatingthe progress of) or remedying a neurodegenerative disease in a patientdiagnosed with the neurodegenerative disease.

In one embodiment, the treatment method of the subject invention reducestau levels and/or improves tau clearance. Normal, functional tau is lessaffected by clearance pathways in the cell than aberrant tau. In oneembodiment, the treatment method of the subject invention modulates tauclearance by selectively targeting abnormal tau.

In an embodiment, the subject invention provides a method for treatingor ameliorating a neurodegenerative disease or condition. The methodcomprises administering, to a subject in need of such treatment, aneffective amount of compounds and compositions of the subject invention.

In an embodiment, the therapeutic composition is administered to a humansubject who has symptoms of, or is diagnosed with, a neurodegenerativedisease. In preferred embodiments, the therapeutic composition isadministered to a human subject who has symptoms of, or is diagnosedwith, a neurodegenerative disease associated with abnormal accumulationof protein tau. For instance, the therapeutic composition isadministered to a human subject who has elevated levels of solubleprotein tau and/or hyperphosphorylated protein tau in the nervoussystem, such as in the brain or cytoplasm of neuronal and glial cells.In addition, the therapeutic composition is administered to a humansubject who exhibits pathologic features such as neurofibrillary tanglesor senile plaques in neuronal cells and/or cell processes. In a specificembodiment, the therapeutic composition is administered to a humansubject who has symptoms of, or is diagnosed with, Alzheimer's disease.

The identification of subjects who are in need of treatment for aneurodegenerative disease is well within the knowledge and ability ofone skilled in the art. By way of example, a clinician skilled in theart can readily identify, by the use of clinical tests, neurologic andphysical examination, and medical/family history, those patients who aresuffering from a neurodegenerative disease as well as those who arepredisposed to developing a neurodegenerative disease and thus readilydetermine if an individual is in need of treatment of the subjectinvention. For instance, neurofibrillary tangles or senile plaquespresent in neuronal cells and/or cell processes can be determined usingelectron microscopy (EM) or other clinical techniques known in the art.In addition, spinal fluid or cerebral fluid samples or tissues samplesfrom hippocampal tissue or frontal cortex tissue samples may be obtainedfrom a subject and levels of protein tau present in the samples can bedetermined using routine techniques such as enzyme-linked immunosorbantassay (ELISA), western blot, and immunological assays.

The term “effective amount” or “therapeutically effective amount,” asused herein, refers to an amount that is capable of preventing, treatingor ameliorating a disease or condition or otherwise capable of producingan intended therapeutic effect. For instance, the effective amount ofthe compounds and compositions of the subject invention is an amountcapable of reducing levels of protein tau in a subject. In certainembodiments, the effective amount enables a 5%, 25%, 50%, 75%, 90%, 95%,99% and 100% reduction of levels of protein tau (e.g. soluble proteintau intermediates and/or aberrant protein tau) in a subject.

The compounds and compositions of the subject invention can be used totreat or ameliorate neurodegenerative diseases including, but notlimited to, Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis (ALS), Huntington's disease, multiple sclerosis,Pick's disease, fronto temporal dementia, cortico-basal degeneration,progressive supranuclear palsy, Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, and Kuru.

The compounds and compositions of the subject invention can also be usedto treat or ameliorate neurodegenerative diseases including, but notlimited to, Down's syndrome, Argyrophilic grain disease, parkinsonismdementia complex of Guam, non-Guamanian motor neurone disease with NFT,Niemann-Pick disease type C, subacute sclerosing panencephalitis,postencephalitic parkinsonism, dementia pugilistica, myotonic dystrophy,prion protein amyloid antipathy, and Hallervorden-Spatz disease.

The compounds and compositions of the subject invention are particularlyuseful to treat or ameliorate a neurodegenerative disease involving taupathologies (i.e., tauopathies) including, but not limited to,Alzheimer's disease, Parkinson's disease, frontotemporal dementia,frontotemporal dementia with Parkinsonism, frontotemporal lobe dementia,pallidopontonigral degeneration, progressive supranuclear palsy,multiple system tauopathy, multiple system tauopathy with preseniledementia, Wilhelmsen-Lynch disease, Pick's disease, and Pick'sdisease-like dementia.

Specifically, the compounds and compositions of the subject inventionare particularly useful to treat or ameliorate a disease or conditionarising, at least in part, from abnormally high levels of protein tau inthe nervous system, such as in cytoplasm of neuronal and glial cells andin neuronal and glial cell processes. Thus, the subject invention isparticularly useful for treatment of neurodegenerative diseases anddisorders, in which reduction of levels of protein tau in the nervoussystem would be beneficial.

In addition, the compounds and compositions of the subject invention areuseful for alleviating or attenuating symptoms arising from orassociated with neurodegenerative diseases, including cognitivedysfunction, impaired memory, impaired mental capacities, emotionaldisturbances, speech dysfunction, incontinence, tremor, posturalinstability, rigidity or stiff movement, muscle paralysis, and pain.

Therapeutic Compositions and Formulations

The subject invention further provides therapeutic compositions thatcontain a therapeutically effective amount of the compounds andcompositions and a pharmaceutically acceptable carrier or adjuvant.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as used herein, include compositions,carriers, diluents and reagents, are used interchangeably, and representthat the materials are capable of administration to or upon a subjectsuch as mammal.

The term “carrier” refers to an adjuvant, excipient, or vehicle withwhich the compound is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleumoil such as mineral oil, vegetable oil such as peanut oil, soybean oil,and sesame oil, animal oil, or oil of synthetic origin. Particularlypreferred pharmaceutical carriers for treatment of or amelioration of aneurodegenerative disease are carriers that can penetrate theblood/brain barrier.

Suitable carriers also include ethanol, dimethyl sulfoxide, glycerol,silica, alumina, starch, sorbitol, inosital, xylitol, D-xylose,mannitol, powdered cellulose, microcrystalline cellulose, talc,colloidal silicon dioxide, calcium carbonate, calcium phosphate, calciumaluminium silicate, aluminium hydroxide, sodium starch phosphate,lecithin, and equivalent carriers and diluents. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidcarriers, particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The therapeuticcomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary, depending on thetype of the condition and the subject to be treated. In general, atherapeutic composition contains from about 5% to about 95% activeingredient (w/w). More specifically, a therapeutic composition containsfrom about 20% (w/w) to about 80% or about 30% to about 70% activeingredient (w/w).

The compounds and compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive compound(s) iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition.

The therapeutic or pharmaceutical compositions of the subject inventioncan also be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions; however, solid forms suitable for solution, or suspensions,in liquid prior to use also can be prepared. The preparation also can beemulsified, such as oil-in-water emulsion.

The compounds and compositions of the subject invention in prescriptionamounts can be readily made into any form of drug, suitable foradministering to humans or animals. Suitable forms include, for example,tinctures, decoctions, and dry extracts. These can be taken orally,applied through venous injection mucous membranes or inhalation. Theactive ingredient can also be formulated into capsules, powder, pallets,pastille, suppositories, oral solutions, pasteurized gastroentericsuspension injections, small or large amounts of injection, frozenpowder injections, pasteurized powder injections and the like.

Routes of Administration

The compounds and compositions of the subject invention can beadministered to the subject being treated by standard routes, includingoral, inhalation, or parenteral administration including intravenous,subcutaneous, topical, transdermal, intradermal, transmucosal,intraperitoneal, intramuscular, intracapsular, intraorbital,intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, epidural and intrasternalinjection, infusion, and electroporation, as well as co-administrationas a component of any medical device or object to be inserted(temporarily or permanently) into a subject. In a preferred embodiment,the compounds and compositions of the subject invention are administeredorally.

The amount of the therapeutic or pharmaceutical composition of thesubject invention which is effective in the treatment of aneurodegenerative disease will depend on the nature of the disease,condition or disorder and can be determined by standard clinicaltechniques. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

Once improvement of the patient's condition has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, may be reduced as a function ofthe symptoms to a level at which the improved condition is retained.When the symptoms have been alleviated to the desired level, treatmentshould cease. Patients may however require intermittent treatment on along-term basis upon any recurrence of disease symptoms.

In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease, condition or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

EXAMPLE

Following is an example that illustrates procedures for practicing theinvention. The example should not be construed as limiting.

Example 1 Myricanol Derivatives and Uses Thereof for Reducing TauProtein Levels

This Example shows myricanol derivatives, their synthetic schemes, anduses for reducing protein tau levels.

(+)-aR,11S-Myricanol Extraction from Bayberry Root Bark Myrica cerifera(Natural Frontier Co-Op)

Tau-reducing-guided fractionation study of Myrica cerifera (bay berryroot bark) led to the identification (+)-aR,11S-myricanol as a taudestabilizer agent with an IC₅₀ of 35 μM. The bayberry root-bark powder(5×1 kg) was extracted with 5×2 L of toluene under agitation in a (5×)flask protected from light (3×24 h). The filtrate was concentrated andgave 58 g of extract. The toluene extract was purified using silica gelMPLC, eluting with a gradient of 0 to 40% ethyl acetate/hexanes to yield27 g of (+)-aR,11S-myricanol [α]_(D) ²¹=48.2 (c=1.1, Chloroform).

Derivatization

Oxidation

Myricanol (120 mg) was treated in a solution of dichloromethane (10 mL)with 150 mg of pyridinium chlorochromate (PCC). After 2 h the reactionwas treated into ice-water and EtOAc extraction. The organic layer wasthen extracted with saturated aqueous NaHCO₃ and brine, then dried overMgSO₄ powder and filtered. The residue was then purified by normal phaseHPLC using an isocratic solvent mixture of 20% ethyl acetate and 80%hexane to give 20 mg of myricanone.

Dehydration

Myricanol (50 mg) was treated in a solution of 10 mL of toluene with 79mg of para toluene sulfonic acid. The solution was boiled for 24 h,cooled, diluted with hexane, washed successively with saturatedsolutions of NaHCO₃ and NaCl, dried over MgSO₄, and evaporated. Themixture was purified by a first HPLC using normal phase column in anisocratic solvent mixture of 3% ethyl acetate/hexane. Two fractions werecollected. The first eluted fraction was purified by reversed phase HPLCusing an isocratic solvent mixture of 32% acetonitrile/water to give 5mg of pure myricacene A (trans) material and 0.5 mg of myricacene A(cis) and 2 mg of myricacene B (trans). The second fraction wasseparated using the same reverse phase condition and provided 21 mg and8 mg of the respective 6-membered-ring and 5-membered ring derivatives[α]_(D) ²¹=0 (c=0.1, Chloroform).

When para toluene sulfonic acid was added at catalytic level (0.3 mg,during 72 hours) after normal and reverse phase HPLC purification: 15 mgof myricacene A (trans), 9 mg of myricacene A (trans), 4 mg ofmyricacene A (cis), 4 mg of myricacene B (cis), 3 mg of the 6-memberedring derivative and 3 mg of the 5-membered ring derivative.

Each of the 6-membered ring and the 5-membered ring derivatives (shownabove) have four possible isomers by the chiral center at position C12and the biphenyl orientation.

During the purification process, the 6-membered ring provided crystaland the X-ray crystallographic analysis provided the relativeconfiguration below:

Methylation

1) Sodium hydride (4 mg) was added to a solution of myricanol (20 mg) inmethyl iodide (1 ml) and dimethylacetamide (DMA, 5 ml) at 0° C. for 30min. The reaction mixture was kept for 4 h at 0° C. then diluted withwater and extracted with diethyl ether. The ether extract was washedwith water and brine, and was dried over sodium sulfate, filtered andevaporated. The residue was separated by normal phase HPLC using 45%ethyl acetate and 55% hexane and provided 8 mg of trimethyl myricanoland 5 mg of dimethylmyricanol.

2) 10 mg of myricacene A (trans) was methylated using the same reactioncondition and treatment process as the previous reaction. After normalphase HPLC using 25% ethyl acetate and 75% hexane the reaction gave 5 mgof dimethylmyricanone A (trans).

Demethylation

10 mg of myricanol was mixed to 5 mL of dichloromethane (3 mL) then theresulting solution was added to solution of 5 mL dichloromethane and 0.2mL BBr₃ at −10° C. and stirred under nitrogen for 1 h to roomtemperature. The reaction mixture was diluted with water, stirred andextracted with chloroform. The chloroform extract was washed with brine,dried over magnesium sulfate, filtered and evaporated to give afterreverse phase HPLC (65% water and 35% acetonitrile) 2 mg of3-hydroxy-myricanol, 3 mg of 4-hydroxy-miricanol and 1 mg of3,4-dihydroxy-myricanol.

Ozonolysis

1) 3 mg of myricacene A (trans) was added to 5 mL of dichloromethane (3mL) then under an ozone generator during 30 min. The reaction mixturewas then treated by dimethyl sulfide for 1 h. The reaction mixture wasevaporated and provided 1 mg of dialdehyde A.

2) The same condition as the previous reaction was applied to 3 mg ofmyricacene B (trans) and gave 0.5 mg dialdehyde A.

Esterification

1) Myricanol (100 mg) was treated with a solution of acetone andpalmitoyl chloride (127 μL) with potassium carbonate (193 mg) at roomtemperature for 15 h. The mixture was then concentrated in Vacuo andfractionated on a silica gel column using MPLC (silica cartridge, IscoCombiflash) with a linear gradient of 0-30% ethyl acetate/hexanes.

The major compound eluted was myricanol 11-monopalmitate (21 mg) with anoptical rotation of [α]_(D) ²¹=44.6 (c=0.9, Chloroform).

2) Myricanol (100 mg) was added to a solution of pyridine (5 ml) andchloroform (3 mL) with palmitic anhydride (500 mg) at room temperaturefor 48 h. The mixture was then concentrated in vacuo and fractionated ona silica gel column using MPLC (silica cartridge, Isco Combiflash) witha linear gradient of 0-30% hexane/ethyl Acetate. The major compoundeluted was myricanol 17-palmitate Myricanol 5,17-dipalmitate, myricanol17-palmitate and myricanol 5-palmitate: (+)-S-(60 mg). Myricanol5-palmitate and myricanol 11,17-dipalmitate were purified usingsemi-preparative column for normal phase HPLC with a gradient of 0 to20% Ethyl acetate hexane providing respectively 4 and 6 mg.

3) Myricacene B (E) (10 mg) was added to a solution of pyridine (5 ml)and chloroform (3 mL) with palmitic anhydride (50 mg) at roomtemperature for 48 h. The mixture was then concentrated in Vacuo andfractionated on a silica gel column using HPLC a linear gradient of0-30% Hexane/Ethyl Acetate. The major compound eluted was myricacene B(trans) 5 palmitate,

Etherification

The 6-membered-ring derivative (5 mg) and 1 mg of 18-crown-6 were addedto a solution of acetonitrile and 1-bromohexadecane (50 μL) withpotassium carbonate (193 mg) at room temperature for 24 h. The mixturewas diluted with water and diethyl ether, washed with brine. The organiclayer was dried over MgSO₄, filtered and evaporated. The residue wasseparated on gradient HPLC (0-30% Ethyl acetate in hexane) to afford 2mg of dihexadecacyloxy 6-membered ring derivative.

To assess the effects of the aqueous bayberry extract on overexpressedand endogenous tau levels, HeLa cells stably transfected with tau weretreated with the myricanol derivatives.

Molecular Compound Structure Weight Name/sample names Results 9

338.3539 Demethoxycurcumin active 10

308.3279 Bisdemethoxycurcumin active 11

350.4923 [10]-Gingerol active 18

340.4129 Myricacene A (trans)/ Myr-R-9-4; Myr-16-13-4 active 19

340.4129 Myricacene B (trans)/ Myr-R-9-2-8; Myr-R-16-15 active 21

340.4129 Myricacene A (cis)/ Myr-R-9-2-4; Myr-R-16-13-2 moderatelyactive 22

340.4129 Myricacene B (cis)/ Myr-R-9-2-6 active 23

340.4129 Myr-R-16-6 very active 24

340.4129 Myr-R-16-4-2 active

The results show that myricanol derivatives myricacene B (cis) (Compound22) and the 6-membered ring (Compound 23) potently reduce levels of tauproteins, and thus, can be used to treat tauopathies such as Alzheimer'sdisease.

Time-dependent bioassay revealed that the 6-membered-ring derivative(Compound 23) potently reduces protein tau levels upon treatment of HeLacells for 24 hours. Dose-dependent bioassay revealed that myricacene B(cis) (Compound 22) is a potent tau-reducing compound at a concentrationof 20 μg/mL. The active synthesized compounds exhibit low toxicity anddo not alter the amount of glyceraldehyde 3-phosphate dehydrogenase(GADPH) levels (they also showed an IC₅₀ against A549>10 μg/mL).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

We claim:
 1. A method of reducing intracellular tau levels, wherein saidmethod comprises administering, to cells comprising protein tau, aneffective amount of a compound of formula A, formula C to formula F, orformula J to formula L:

wherein each of R₁-R₄ of formula A, formula C, formula D, formula F,formula J, formula K, and formula L is, independently, —H or any groupthat forms an ester or ether bond; wherein each of R₁-R₈ of formula Eis, independently, H or any group that forms an ester or ether bond;whereby intracellular tau level is reduced.
 2. The method according toclaim 1, wherein the compound has a chemical structure of formula A. 3.The method according to claim 1, wherein the compound has a chemicalstructure of formula C.
 4. The method according to claim 1, wherein thecompound is Compound 22, or Compound 24:


5. The method according to claim 1, wherein the cells are in a subjectin need of treatment for a neurodegenerative disease.
 6. The method ofclaim 5, wherein the neurodegenerative disease is a tauopathy.
 7. Themethod of claim 6, wherein the subject is a human.
 8. The method ofclaim 7, wherein the neurodegenerative disease is selected fromAlzheimer's disease, Parkinson's disease, frontotemporal dementia,frontotemporal dementia with Parkinsonism, frontotemporal lobe dementia,pallidopontonigral degeneration, progressive supranuclear palsy,multiple system tauopathy, multiple system tauopathy with preseniledementia, Wilhelmsen-Lynch disease, Pick's disease, and Pick'sdisease-like dementia.
 9. The method of claim 8, wherein theneurodegenerative disease is Alzheimer's disease.